Extracellular signals such as hormones and cytokines modulate many cellular processes by activating adenylate cyclase, increasing intracellular levels of cAMP and ultimately activating the cAMP-dependent kinase (PKA). PKA is a ubiquitous enzyme that functions in many intracellular pathways, for example, regulation of glycogen metabolism by reversible phosphorylation of glycogen phosphorylase [Walsh et al., J. Biol. Chem., 243:3763-3765 (1969)], and regulation of MAP kinase signaling by inhibiting Raf-1 activation by Ras [Vojtek et al., Cell, 74:205-214 (1993) and Hafner et al., Mol. Cell Biol., 14:6696-6703 (1994)]. Inactive PKA exists as a tetramer in which two identical catalytic subunits are bound to a dimer of two regulatory subunits. Activation of PKA by cAMP is effected by binding of a cAMP molecule to each of the regulatory subunits (R) causing release of the active catalytic subunit (C). While only one form of the C subunit has been identified, two classes of R subunit exist, RI and RII, with apparently distinct subcellular distributions. The RI isoforms (RI.alpha. and RI.beta.) are reported to be predominantly cytoplasmic and are excluded from the nucleus, whereas up to 75% of the RII isoforms (RII.alpha. or RII.beta.) are particulate and associated with either the plasma membrane, cytoskeletal components, secretory granules, golgi apparatuses, centrosomes or possibly nuclei [Scott, Pharmac. Ther., 50:123-145 (1991)]. Presumably, differences (either physical or physiological) in the various R subunits provide a means by which cells are able to restrict activity of the C subunit to a desired pathway.
Recent evidence indicates that cells are able to target PKA activity by localizing the inactive enzyme in the vicinity of potential substrates, thereby restricting the activity of the C subunit following release by cAMP binding to the R subunit. This "compartmentalization" segregates PKA with participants in a given signaling pathway and contributes to PKA specificity in response to different extracellular stimuli. Compartmentalization of PKA occurs, at least in part, by interaction or tethering, of the R subunit with specific proteins which localize, or anchor, the inactive holoenzyme at specific intracellular sites. Proteins which specifically segregate PKA have been designated A Kinase Anchor Proteins, or AKAPs [Hirsch et al., J. Biol. Chem., 267:2131-2134 (1992)]. In view of the fact that some AKAP have been shown to bind, and anchor, other proteins in addition to PKA, the family of proteins is generally referred to as anchoring proteins.
To date, a number of anchoring proteins have been identified [discussed below] which apparently bind PKA by a common carboxy terminal secondary structure motif that includes an amphipathic helix region [Scott and McCartney, Mol. Endo., 8:5-11 (1994)]. Binding of PKA to most, if not all, identified anchoring proteins can be blocked in the presence of a peptide (Ht31) that mimics this common secondary helical structure, while a mutant Ht31 peptide, containing a single animo acid substitution that disrupts the helical nature of the peptide, has no effect on PKA/anchoring protein binding [Carr et al., J. Biol. Chem., 266:14188-14192 (1991)]. Even though PKA/anchoring protein interaction is effected by a common secondary structure, anchoring proteins (or homologous anchoring proteins found in different species) generally have unique primary structure as evidenced by the growing number of anchoring proteins that have been identified in a variety of organisms. Presumably, the unique amino acid structure, most notable in amino terminal regions of the proteins, accounts in part for anchoring proteins identified as unique to various specific cell types and for the various specific intracellular compartments in which PKA localization has been observed.
For example, anchoring proteins which are predominantly expressed in mammalian brain have been identified in the rat (AKAP 150) and cow (AKAP 75) [Bergman, et al., J. Biol. Chem. 266:7207-7213 (1991)], as well as in humans (AKAP 79) [Carr, et al., J. Bio. Chem. 267:16816-16823 (1992)]. Amino acid identity and immunological cross-reactivity between these neuronal-specific proteins suggest that they represent interspecies homologs. As another example, AKAP 100 appears to be specific for human and rat cardiac and skeletal muscle, while being expressed to a lower degree in brain cells of these mammals. As still another example, AKAP Ht31 [Carr et al., J. Biol. Chem., 267:13376-13382 (1992)] appears to be specific for thyroid cells. Conversely, AKAP 95 has been shown to be expressed in a multitude of cell types, showing no apparent tissue or cell-type specificity.
With regard to localization in specific intracellular compartments, AKAP 75, microtubule-associated protein (MAP-2) [Threurkauf and Vallee, J. Biol. Chem., 257:3284-3290 (1982) and DeCamilli et al., J. Cell Biol., 103:189-203 (1986)], AKAP 79 [Glantz et al., J. Biol. Chem., 268:12796-12804 (1993)] and AKAP 150 [Glantz et al., Mol. Biol. Cell, 3:1215-1228 (1992)] are closely associated with cytoskeletal structural proteins, with AKAP 75 more specifically associated with post synaptic densities [Carr et al., J. Biol. Chem., 267:16816-16823 (1992)]. Still other anchoring proteins have been shown to localize with less widespread cellular structures, including AKAP 350 association with centrosomes [Keryer et al., Exp. Cell Res., 204:230-240 (1993)], AKAP 100 with the sarcoplasmic reticulum in rat cardiac tissue [McCartney, et al., J. Biol. Chem. 270:9327-9333 (1995)], and an 85 kDa AKAP which links PKA to the Golgi apparatus [Rios et al., EMBO J., 11:1723-1731 (1992)].
AKAP 95, with an apparent zinc finger DNA-binding region, appears to reside exclusively in the nucleus [Coghlan et al., J. Biol. Chem., 269:7658-7665 (1994)]. The DNA binding domain of AKAP 95 provides a role for direct involvement of PKA in gene transcription, possible by positioning of PKA for phosphorylation of transcription factors. Other diverse cellular activities shown to be influenced by anchoring protein/PKA binding have been demonstrated by disruption of the interaction, for example, disruption of PKA/anchoring protein binding in T cells has been shown to reverse cAMP-induced suppression of interleukin 2 expression [Lockerbie et al., J. Cell Biochem., Suppl. 21A:76, Abstract D2155 (1995)] and disruption of PKA/anchoring protein binding in hippocampal neurons has been shown to attenuate whole cell currents through alpha-amino-3-hydroxy-5-methyl-4isoxazole propionic acid/kainate glutamate receptors [Rosenmund et al., supra.]. The ability of anchoring proteins to regulate IL-2 expression and to regulate glutamate receptor activity, in combination with a previous demonstration that anchoring proteins can bind calcineurin, suggest multiple therapeutic applications for anchoring proteins and molecules which modulate anchoring protein binding to cellular components.
In view of the diversity, both in terms of cell type expression, subcellular localization and physiological activities of anchoring proteins identified to date, there thus exists a need in the art to continue to identify novel anchoring proteins and nucleic acids which encode them. The uniqueness of anchoring protein primary structures provides a target for specifically regulating PKA localization, and thereby its function in specific cellular processes.